T lymphocytes, also referred to in the art as T cells, are known to mediate immune responses in at least two distinct ways. Cytotoxic T cells are involved in the lysis of specific target cells, while helper T cells assist in the proliferation and differentiation of B cells, leading to the production of antigen-specific antibodies [see, e.g., Kimball (ed.), Introduction To Immunology, 2nd Ed. , Chapters 11-13, Macmillan Publishing Co. (1986)]. Either type of T cell is activated to perform its function via the interaction of the T cell receptor (Ti) or its associated CD3 complex (also referred to as the CD3/Ti receptor complex) on the T cell surface with an antigen on an antigen-presenting cell [see, e.g., Reinherz et al., "Clonotypic Surface Structure On Human T Lymphocytes: Functional And Biochemical Analysis Of The Antigen Receptor Complex", Immunol. Rev., 81:95-129 (1984)].
It has been found, however, that T cells respond to an antigen only when that antigen is associated with a particular MHC (major histocompatibility complex)-encoded antigen on the antigen-presenting cell. T cell recognition of an antigen is said therefore to be restricted by the class of MHC-encoded product associated with the antigen. The result of this MHC restriction is that cytotoxic T cells are activated by antigens in association with class I MHC antigens and helper T cells are activated by antigens associated with class II MHC antigens [see, e.g., Kimball (ed.), Introduction To Immunology, supra at pp. 286-89 and pp. 348-58].
Furthermore, it has been recognized that the class II MHC-restricted T cells express, in addition to the CD3/Ti receptor complex, a cell surface antigen termed CD4, whereas the class I MHC-restricted T cells express a CD8 cell surface antigen. This association between MHC restriction and CD antigen expression has led to the hypothesis that, in T cell activation, the respective MHC antigens are the natural ligands for the CD antigens, i.e., the CD4 antigen on helper T cells interacts with the class II MHC molecules on the antigen-presenting cells (e.g., macrophage) and the CD8 antigen on cytotoxic T cells interacts with the class I MHC molecules on specific target cells (e.g., virus-infected cells) [see, e.g., Emmrich et al., "Cross-Linking Of The T Cell Receptor Complex With The Subset-Specific Differentiation Antigen Stimulates Interleukin 2 Receptor Expression In Human CD4 And CD8 T Cells", Eur. J. Immunol., 17:529-34 (1987)]. In addition, it has been proposed that the recognition of antigen by T cells may occur via a quaternary complex wherein the CD3/Ti receptor complex on the T cell surface, in association with a CD antigen such as CD4 or CD8, "sees" the antigen in association with the appropriate MHC-encoded antigen [see, e.g., Anderson et al. , "Cross-Linking Of T3 (CD3) With T4 (CD4) Enhances The Proliferation Of Resting T Lymphocytes", J. Immunol., 139 (No. 3):678-82 (1987)].
As used in this application, CD antigens are naturally-occurring cell surface differentiation antigens defined by the reactivity of monoclonal antibodies (mAbs) on the surface of cells, as designated by an International Workshop whose function is to provide a unified nomenclature for these antigens [see, e.g., McMichael (ed.), Leukocyte Typing III, Oxford University Press (Oxford, U.K. 1987)]. A large number of CD antigens have been molecularly cloned and are therefore fully characterized [Id.]. Well known CD antigens include the pan-T antigens, CD3, CD2, CD5, CD6 and CD7, a CD antigen specific for the helper T cell subset, CD4, and a CD antigen specific for the suppressor T cell subset, CD8 [see, e.g., Ledbetter et al., in Perspectives In Immunogenetics And Histocompatibility, Vol. 6, E. Heise (ed.), Lymphocyte Surface Antigens 1984, pp. 325-40, American Society For Histocompatibility And Immunogenetics (New York 1984)]. CD28 is an antigen present on a majority of T cells [Yamada et al., Eur. J. Immunol. 15:1164-1169 (1985)]. Although these differentiation antigens are thought to function as receptors as described above and have been linked to signal transduction in T cells, their exact function in T cell activation is not known and is the subject of intense research.
For example, studies have been carried out to determine the role of CD antigens in T cell activation on a molecular level. It is known that the reaction of an CD3/Ti receptor complex with either mAb or antigen leads to the generation of a transmembrane "signal" within the T cell. This signal is often characterized by the production of inositol monophosphate, bisphosphate and trisphosphate, and an increase in the concentration of cytoplasmic free calcium ([Ca.sup.2+ ].sub.i) [see, e.g., Imboden et al., "Transmembrane Signalling By The T Cell Antigen Receptor", J. Exp. Med., 161:446-56 (1985) and Imboden et al., "Antigen Recognition By A Human T Cell Clone Leads To Increases In Inositol Triphosphate", J. Immunol., 103 (No. 5):1322-24 (1987)].
The generation of this signal may not be sufficient, however, to stimulate the T cell to proliferate [Id. at 873]. Under experimental conditions, when the CD3 T cell surface antigen is cross-linked, e.g., by reacting the cell with a cross-linked antibody, the antibody having been cross-linked either in the presence of accessory cells (e.g., by the interaction of the monocyte Fc receptor with the Fc portion of an antibody to CD3) or by immobilization of the antibody on a solid support, T cell proliferation results.
Thus, mAbs to the CD3/Ti receptor complex that have been coupled to a solid support stimulate T cells to proliferate [see, e.g. Anderson et al., supra and Walker et al., "Activation Of T Cells By Cross-Linking An Anti-CD3 Antibody With A Second Anti-T Cell Antibody: Mechanism And Subset-Specific Activation", Eur. J. Immunol., 17:873-80 (1987)], in part due to the cross-linking of the CD3 antigen and in part due to the prevention of internalization of the CD3/Ti receptor complex, because internalization tends to inhibit T cell activation [see, e.g., Ledbetter et al., "Valency Of CD3 Binding And Internalization Of The CD3 Cell-Surface Complex Control T Cell Responses To Second Signals", J. Immunol., 136:3945-52 (1986)]. There are, however, substantial difficulties with the use of an anti-CD3 immobilized on a solid support to enhance T cell activation in vivo for therapeutic applications. Such a use would involve injection of the solid support, usually small beads, into a patient which would entail certain dangers to the health of the patient, such as blocked or clogged arteries.
Furthermore, it has been proposed that under physiological conditions, even this CD3/Ti stimulation via cross-linking of the CD3 antigen exerts only a minimal signal which may require enhancement by interactions on the T cell surface between CD3/Ti and other CD antigens [see Anderson et al., supra at p.678].
In addition, there is uncertainty as to the exact effect of CD antigen interactions in T cell activation. For example, analysis of spontaneous CD4 loss variants [see Marrack et al., "The Major Histocompatibility Complex Restricted Antigen Receptor On T Cells II. Role Of The L3T4 Product", J. Exp. Med., 158:1077-91 (1983)], as well as gene transfer experiments [see, e.g., Gay et al., "Functional Interaction Between Human T-Cell Protein CD4 And The Major Histocompatibility Complex HLA-DR Antigen", Nature, 328:626-29 (1987)], suggest that the expression of CD4 augments T cell responses to specific antigen and that CD4 plays a particularly important role when concentrations of antigens are suboptimal or when the avidity of the T cell for antigen/MHC-encoded antigen is low [see, Regnier-Bigouroux et al., "Accessory Molecules And T Cell Activation I. Antigen Receptor Avidity Differentially Influences T Cell Sensitivity To Inhibition By Monoclonal Antibodies To LFA-1 And L3T4", Eur. J. Immunol., 16:1385-90 (1986) and Shaw et al., "Susceptibility Of Cytotoxic T Lymphocyte (CTL) Clones To Inhibition By Anti-T3 And Anti-T4 (But Not Anti-LFA-1) Monoclonal Antibodies Varies With The `Avidity` Of CTL-Target Interaction", J. Immunol., 134 (No. 5):3019-26 (1985)]. Furthermore, when mAbs reactive with CD3 and CD4 were immobilized on the same solid support, proliferation of T cells exposed to the immobilized antibodies was enhanced. Similarly, when mAbs to CD3 and CD8 were coupled to a solid support, proliferation of T cells was enhanced over that observed upon exposure to antibody to immobilized CD3 alone [see, Anderson et al., supra.]
However, studies with soluble CD4 monoclonal antibody indicate that the soluble antibody inhibits T cell responses, suggesting that CD4 may transmit a negative signal that inhibits T cell activation [see, e.g., Bank et al., J. Exp. Med., 163:1294 (1985); Tite et al., "The Role Of L3T4 In T Cell Activation: L3T4 May Be Both An Ia-Binding Protein And A Receptor That Transduces A Negative Signal", J. Cell. Mol. Immunol., 2:179-90 (1986); and Rosoff et al., "The Role Of The L3T4 Molecule In Mitogen And Antigen-Activated Signal Transduction", Cell, 49:845-53 (1987)].
B lymphocytes, also known as B cells, are the precursors of antibody-producing (plasma) cells. When B cells are stimulated by an antigen, requiring the cooperation of helper T cells and macrophages, the B cells proliferate and differentiate into plasma cells and memory B cells. CD antigens that have been identified on B cells include CD19, CD20, CDw4O, CD45, CD45R and Bgp95 (an antigen that has not yet received a CD designation) [McMichael, Leukocyte Typing III, supra].
Another CD antigen of interest is the CD45 leukocyte common antigen (L-CA, also known as T200 or Ly-51. CD45 encompasses a family of major glycoproteins ranging from a molecular weight (Mr) of 180 to 220 kDa that is restricted to cells of hematopoietic lineages [Trowbridge "et al., Proc. Nat'l. Acad. Sci. USA 72:157-161 (1975); Komuro et al., Immunogenetics 1:452-456 (1975); Schied et al., Immunogenetics 9:423-433 (1979) Dalchau et al., Eur. J. Immunol. 10:737-744 (1980); and Omary et al., J. Exp. Med. 152:842-852 (1980)]. Distinct isoforms of CD45 arise from alternative mRNA splicing. These isoforms are differentially expressed on subpopulations of T and B lymphocytes. Some monoclonal antibodies (mAbs) to human CD45 antigen recognize epitopes shared by all CD45 isoforms of Mr 220, 205, 190 and 180 kDa. [Cobbold et al., Leukocyte Typing III, supra, Chap. 15, pp 788-803). However, other mAbs recognize only the 220 kDa isoform of CD45, designated CD45R, that is selectively expressed on B lymphocytes and a subpopulation of T cells. [Cobbold, supra; Dalchau et al., J. Exp. Med., 153:753-765 (1981); Morimoto et al., J. Immunol. 134:1508-1515 (1985); and Ledbetter et al., J. Immunol. 135:1819-1825 (1985)]. Another mAb, UCHL-1, selectively binds to the 180 kDa species, which is restricted to cortical thymocytes and a subset of activated or memory T cells. [Smith et al., Immunology 58:63-70 (1986)].
Recently, the primary structures of rat [Thomas et al., Cell, 41:83-93 (1985) and Barclay, et al., EMBO J. 6:1259-1264 (1987)], mouse [Saga, et al., Proc. Natl. Acad. Sci. USA 83:6940-6944 (1986); W. C. Raschke, Proc. Natl. Acad. Sci. USA 84:161-165 (1987); Thomas et al. Proc. Natl. Acad. Sci. USA 84:5360-5363 (1987) and Saga et al., Proc. Natl. Acad. Sci. USA 84:5364-5368 (1987)], and human [Ralph et al., EMBO J. 6:1251-1257 (1987) and Streuli et al., J. Exp. Med. 166:1548-1566 (1987)] CD45 (L-CA) have been deduced from CDNA nucleotide sequences. CD45 is an integral membrane protein with a large 705-707 amino acid cytoplasmic segment, a 22 amino acid trans-membrane segment, and an extracellular domain ranging from 400 to 550 amino acids. The various isoforms of CD45 that are generated by alternative mRNA splicing of primary transcripts of a single gene have different extra-cellular domains, but have the same transmembrane and cytoplasmic segments [Barclay, supra; Ralph, supra and Streuli, supra]. The cytoplasmic segment has two homologous, highly conserved domains of approximately 300 residues.
Studies attempting to define the function of CD45 have yielded conflicting results [Cobbold, suDra]. mAbs to the human or mouse antigen have been reported to inhibit T and B cell proliferation [Harp et al., J. Immunol. 133:10-15 (1984); Bernabeu, et al., Eur. J. Immunol. 17:1461-1466 (1987) and Mittler et al., J. Immunol. 138:3159-3166 (1987)], cellular differentiation [Yakura, J. Exp. Med. 157:1077-1088 (1983)], and natural killer (NK) cell and cytotoxic T cell activity [Seaman et al., J. Immunol. 127:982-986 (1981); Newman, Proc. Nat'l. Acad. Sci. USA 79:3858-3862 (1982); Nakayama et al., Proc. Nat'l. Acad. Sci. USA 76:1977-1981 (1979) and Lefrancois et al., Nature(London) 314:449-452 (1985)]. However, under certain conditions, anti-CD45 mAbs have also been reported to augment T cell proliferation [Cobbold, supra; Ledbetter, supra and Martorell, et al., Eur. J. Immunol. 17:1447-1451 (1987)]. Thomas, supra, have suggested that the 80 kDa cytoplasmic portion of the molecule with its two conserved homologous domains may play a critical role in CD45 function. Recently, these domains were found to be homologous to a major low-molecular weight protein tyrosine phosphatase isolated from both the soluble and particulate fractions of human placenta [Tonks, et al., J. Biol. Chem., 263:6722-6730 (1988); and Charbonneau et al., Proc. Nat'l. Acad. Sci. USA 85:7182-7186 (1988)]. This suggests that CD45 is a membrane-bound protein tyrosine phosphatase which may function by interacting with other membrane-associated molecules.
In the present invention, the antibody heteroconjugates may be presented to react with antigens on lymphocytes to regulate lymphocyte activity. Alternatively, bispecific antibodies, i.e. antibodies containing binding regions reactive with two different cell surface CD antigens may be used to regulate lymphocyte activity.